Daphnia Pulex Lineages in Response to Acute Copper Exposure Sneha Suresh1,2, Teresa J

Suresh et al. BMC Genomics (2020) 21:433 https://doi.org/10.1186/s12864-020-06831-4

RESEARCH ARTICLE Open Access Alternative splicing is highly variable among Daphnia pulex lineages in response to acute copper exposure Sneha Suresh1,2, Teresa J. Crease3, Melania E. Cristescu4 and Frédéric J. J. Chain1*

Abstract Background: Despite being one of the primary mechanisms of gene expression regulation in eukaryotes, alternative splicing is often overlooked in ecotoxicogenomic studies. The process of alternative splicing facilitates the production of multiple mRNA isoforms from a single gene thereby greatly increasing the diversity of the transcriptome and proteome. This process can be important in enabling the organism to cope with stressful conditions. Accurate identification of splice sites using RNA sequencing requires alignment to independent exonic positions within the genome, presenting bioinformatic challenges, particularly when using short read data. Although technological advances allow for the detection of splicing patterns on a genome-wide scale, very little is known about the extent of intraspecies variation in splicing patterns, particularly in response to environmental stressors. In this study, we used RNA-sequencing to study the molecular responses to acute copper exposure in three lineages of Daphnia pulex by focusing on the contribution of alternative splicing in addition to gene expression responses. Results: By comparing the overall gene expression and splicing patterns among all 15 copper-exposed samples and 6 controls, we identified 588 differentially expressed (DE) genes and 16 differentially spliced (DS) genes. Most of the DS genes (13) were not found to be DE, suggesting unique transcriptional regulation in response to copper that went unnoticed with conventional DE analysis. To understand the influence of genetic background on gene expression and alternative splicing responses to Cu, each of the three lineages was analyzed separately. In contrast to the overall analysis, each lineage had a higher proportion of unique DS genes than DE genes suggesting that genetic background has a larger influence on DS than on DE. Gene Ontology analysis revealed that some pathways involved in stress response were jointly regulated by DS and DE genes while others were regulated by only transcription or only splicing. Conclusions: Our findings suggest an important role for alternative splicing in shaping transcriptome diversity in response to metal exposure in Daphnia, highlighting the importance of integrating splicing analyses with gene expression surveys to characterize molecular pathways in evolutionary and environmental studies. Keywords: Splicing, Copper, Metal pollution, Transcriptomics, Daphnia pulex, RNA-seq

* Correspondence: [email protected] 1Department of Biological Sciences, University of Massachusetts Lowell, Lowell, MA 01854, USA Full list of author information is available at the end of the article

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Background transcript thereby regulating the amount of functional Anthropogenic activities such as mining and intensive transcript present in the cell [18]. agriculture have led to a substantial amount of heavy Identification of alternative splicing events from RNA- metal pollution in aquatic ecosystems [1]. Metal contam- seq data involves mapping reads to a reference genome ination poses a great threat to the overall health and sur- to identify splice junctions and counting the number of vival of aquatic organisms due to the long persistence reads aligning to particular exons and splice sites. Per- and bioaccumulation [2]. Exposure to environmental cent Spliced In (PSI) is a widely used metric for quanti- contaminants can induce genomic responses in organ- fying alternative splicing events and detecting differential isms affecting reproduction and survival [3]. In aquatic splicing between conditions. It represents the percentage invertebrates, exposure to metals such as copper has of transcripts including a particular exon or splice site been associated with increased production of reactive and is calculated from the read counts [19]. Unlike gene oxygen species, depletion of glutathione, inhibition of expression analysis where a read falling anywhere along oxidative phosphorylation and antioxidant systems, the gene will count towards expression, identification of DNA damage and inhibition of DNA repair mechanisms a splicing event requires the read to span the splice junc- [4]. Heavy metals further modulate the expression level tion and hence detection of differential splicing events of genes that are actively involved in protecting cells could potentially be biased towards more abundant tran- from metal-induced oxidative stress [2]. Recent genome- scripts [20]. Additionally, abundance of the final mRNA wide studies have provided important insights into the transcript is dependent on several factors such as cell molecular basis of transcription in response to metals, type, developmental stage, disease condition, the pres- but much less is known about the contribution of other ence of intronic and exonic enhancers and silencers, the mechanisms that regulate expression via RNA process- expression of various splicing factors, and can change in ing such as alternative splicing. response to external stimuli and cellular stress [21]. Alternative splicing is a regulatory process in eukary- Identification of intron retention events could be par- otes that generates multiple messenger RNAs (mRNAs) ticularly challenging as it can be difficult to distinguish from a single gene by selective removal or retention of true intron retention events from those arising from in- exons and introns from the pre-mRNA transcript [5]. It completely processed transcripts [22]. However, several is one of the primary mechanisms of gene expression computational tools have been developed for analysing regulation that contributes to transcriptional diversity alternative splicing events from RNA-seq data. While and activity in eukaryotes [6]. An extreme example is some tools like DEXSeq [23] rely on reads assigned to the Dscam gene that can generate 38,016 potential exons, others like JunctionSeq [24] and rMATS [25] use mRNA transcripts in Drosophila melanogaster and over reads aligned to both exons and splice junctions and can 13,000 potential mRNA isoforms in Daphnia via alterna- therefore identify differential splicing events even when tive splicing [7, 8]. Global analyses in eukaryotes have re- the exon expression level is consistent across different ported a large variation in the prevalence of alternative conditions [26]. splicing among taxa [9–11], from about 25% of genes in Several studies have reported that alternative splicing Caenorhabditis elegans [12] and 31% of genes in Dros- plays an important role in abiotic stress tolerance in ophila [13], to over 90% of genes in humans [14]. Gen- plants and mammals [27–30], but whether alternative omic architecture has been suggested to play a role in splicing is a common response to stress in aquatic inver- the diversity of observed frequencies of different types of tebrates is not well understood. Analyses of whole tran- alternative splicing [15]. The main types of alternative scriptomes of plants have shown that alternative splicing splicing events include exon skipping (ES), intron reten- regulates the expression level of genes involved in stress tion (IR), alternative 5′ splice site (A5SS), alternative 3′ response pathways and genes encoding the various com- splice site (A3SS) and mutually exclusive exons (MXE) ponents of a spliceosome, which is an RNA-protein [16]. Exon skipping is the most common type of alterna- complex that directs splicing of pre-mRNA transcripts tive splicing in animals, while intron retention events [31]. Recent studies on plants have also shown that abi- occur at high levels in plants, as well as most fungi and otic stresses such as exposure to high temperatures, high protists [11, 17]. Exon skipping, the presence of MXE, salinity or treatment with the plant hormone abscisic A3SS and A5SS within the exons lead to addition or re- acid alters the alternative splicing patterns of several moval of functional domains or changes in the amino or genes and promotes the use of non-canonical splice carboxy terminus of the protein product thereby affect- sites, thereby increasing the transcriptome diversity in ing its activity, localization, stability and function. Intron adverse environmental conditions [30]. Similar studies retention events usually lead to premature stop codons on nematodes and insects suggest that regulation of al- within the transcripts, which results in the formation of ternative splicing events is a key mechanism in mediat- a truncated protein or in most cases degradation of the ing response to stressors, reporting alternative splicing Suresh et al. BMC Genomics (2020) 21:433 Page 3 of 14

mediated regulation of transcriptional activity in re- response to stress [47]. Previous studies have reported sponse to heat/cold stress [32–35]. In addition, a differences among Daphnia clones in tolerance and genome-wide analysis of alternative splicing events in response to various natural and anthropogenic the Pacific oyster, Crassostrea gigas in response to abi- stressors [46–49], but there is a lack of similar studies otic stressors reported that 16% of the oyster protein in response to metal stress. Heavy metal concentra- coding genes undergo alternative splicing and these tions in the environment continue to be a concern genes are enriched in functions related to cellular me- with ongoing industrial activities [50], and copper is tabolism, cell signaling, and post translational protein one of the most common pollutants that is toxic at modifications [36]. There is a lack of similar analyses of high concentrations [51]. Exposure to sub-lethal con- the relative contribution of alternative splicing in regu- centrations of copper is known to significantly impair lating gene expression in most organisms, including reproductive output in D. pulex [52]. Our recent Daphnia pulex, a well-established model organism for study found that most differentially expressed genes ecological genomics. Differential splicing analysis has the between copper-exposed Daphnia and controls were potential to identify functional diversity that is missed by shared among genetic lineages, but each lineage had a differential gene expression analysis alone, and hence few unique genes that changed in expression under can complement differential gene expression in under- copper exposure [53]. Thus, while stress response standing the genes and molecular pathways involved in mechanisms may be largely similar among the mem- stress response. Altering the splicing patterns is a major bers of a species, individual populations may adopt mechanism that can regulate the levels of gene expres- different mechanisms to adapt to environmental per- sion and inhibit protein synthesis by introducing prema- turbations. Investigating the genetic basis of differen- ture stop codons in the mRNA resulting in their tial gene expression and splicing among Daphnia degradation by the mRNA surveillance system [37, 38]. clones can help distinguish common stress response Furthermore, individual variation in alternative splicing pathways from lineage-specific responses to metal patterns have been shown to alter the phenotypic re- exposure. sponse to stress in various organisms, suggesting that In this study, we integrate an analysis of differential splicing can vary between genotypes [39]. Although al- gene expression and differential splicing to identify the ternative splicing seems to play an important role in role of alternative splicing in mediating response to stress response mechanisms, more studies are needed to metal-induced acute stress in Daphnia. We perform identify the extent of splicing upon exposure to stress as these analyses using our previously published RNA-seq well as the variability within species and how it comple- dataset on lineages that originate from three natural ments the transcriptional response. populations, which was used to determine the extent of The micro-crustacean Daphnia pulex is among the similar responses to Cu among lineages [53]. Here, we most common species of water flea inhabiting lakes add new analyses to determine whether differentially and ponds throughout the world. It was the first spliced genes have similar functional enrichment distri- crustacean to have its genome sequenced and is butions and regulate similar biological processes as dif- widely used as a model organism in environmental ferentially expressed genes, and we also use a new toxicity studies [40]. Daphnia has the ability to de- reference genome assembly with refined gene annota- velop distinct alternative phenotypes in response to tions [42]. This work advances our understanding of the environmental cues and has been considered to have biological significance of alternative splicing events in an ecologically responsive genome [41]. Based on the Daphnia and its impact in shaping the transcriptome di- newest genome assembly, D. pulex has a compact versity in response to metal exposure. genome of 156 Mb consisting of 18,440 genes with relatively small introns and small intergenic spaces Results [42]. Previous work has identified that 51% of D. Differential gene expression in response to acute copper pulex genes and 60% of Daphnia magna genes exposure undergo alternative splicing [43]. Daphnia occur in A total of 21 Daphnia RNA-seq samples were used to diverse environments across a wide range of eco- determine transcriptional responses to Cu exposure logical conditions and the populations have a high de- (Fig. 1a). Across all samples, there was an average of 16, gree of genetic variation [44, 45]. Genetic divergence 151 expressed genes (Table S1), and an average read between Daphnia populations could result in varied depth of 498 per gene and 9.9 per coding bp. A total of phenotypic responses to stressors [46]. Consequently, 588 differentially expressed (DE) genes were identified the effects of stressors on monoclonal populations between all 15 samples exposed to acute Cu stress ver- cannot be extrapolated to the species level as genetic- sus all 6 control samples (FDR corrected p-value < 0.05). ally diverse populations will differ in tolerance and DE genes with known functional annotations accounted Suresh et al. BMC Genomics (2020) 21:433 Page 4 of 14

Fig. 1 a: Experimental set-up for investigating the molecular responses of D. pulex to acute Cu exposure. Individuals from three different clonal lineages (D, K, S) were placed in individual tubes in four separate tanks. b: Volcano plot showing gene expression fold change differences between copper-exposed and control samples. The average log2 fold change in gene expression is on the x-axis (positive values are upregulated in copper-exposed samples), and the average negative log10 of FDR-corrected p-values are on the y-axis. Non-differentially expressed genes are in grey (corrected p-value > 0.05), differentially expressed (DE) genes (corrected p-value < 0.05) are in black and DE genes with corrected p-value < 0.01 and log2 fold change > 4 are in blue. The five labelled genes correspond to the annotated DE genes that are strongly responsive to acute Cu exposure in Table 1. The blue dotted lines indicate the cut-off values for the log2fold change and FDR-corrected p-value for the genes identified to be strongly responsive to acute Cu exposure. for 64% (377/588) of all DE genes, similar to the were not DE in the current study. A total of 40 genes genome-wide proportion (63%). Five hundred genes did not have any reciprocal blast hits, probably because were upregulated (log2fc > 0) in response to acute Cu there are multiple duplicate genes in the 2011 genome exposure and 88 genes were downregulated (Fig. 1b; (Tables S3,S4), as suggested by Ye et al. [42]. Table S2). Of the DE genes, 20 genes had an FDR corrected p-value < 0.01 and an absolute log2 fold change > 4, which we identified as having the most extreme ex- Identification of alternative splicing events in Daphnia pression differences between copper and controls Across the three clonal lineages, a total of 6630 of (Fig. 1b). Five of these 20 DE genes are annotated and the 17,761 expressed genes were identified to have al- characterized: metallothionein b (mtb), alpha-carbonic ternative transcripts, accounting for ~ 37% of the D. anhydrase (aca1), vitelline membrane outer layer protein pulex genes (Table S1). Specifically, 4820, 4738 and 1 (vmo1), rna polymerase ii degradation factor 1 (def1), 4721 alternatively spliced (AS) genes were identified and cell recognition protein (caspr4) isoform 5. Six of in Clones K, S and D, respectively. This is slightly these 20 DE genes were also reported as DE in our pre- more than the percentage of alternatively spliced vious study [53] (Table 1), which used the same tran- genes in other species such as C. elegans (25%) [12], scriptomic dataset but the D. pulex genome published in C. gigas (16%) [36]andD. melanogaster (31%) [13], 2011 [41]. We conducted a BLAST analysis to compare but less than a previous estimate of 51% [43]. Five the DE genes identified in this study against the genes AS types were inferred: exon skipping (ES), intron re- from the 2011 genome assembly. Although our previous tention (IR), alternative 5′ splice site (A5SS), alterna- analyses reported in Chain et al. [53] reported three tive 3′ splice site (A3SS) and mutually exclusive times fewer DE genes than we did in this new analysis exons (MXE). The distribution of AS types was simi- (206 genes out of an average of 17,128 expressed genes), lar across the lineages, with A3SS being the most 142 genes out of the 206 genes (69%) were DE in both abundant (52–59%), followed by A5SS (45–50%), IR analyses, and only seven genes with reciprocal blast hits (22–23%), ES (18–19%) and MXE (2%) (Table 2). Suresh et al. BMC Genomics (2020) 21:433 Page 5 of 14

Table 1 Differentially expressed (DE) genes that are strongly responsive to acute Cu exposure. Results are based on the global analysis combining all 6 controls with all 15 Cu exposed samples. Genes that were also reported to be strongly responsive to Cu exposure in a previous study by Chain et al., [53] using the 2011 draft genome are indicated by an asterisk (*) Gene_ID Description blast hit log2 fold change FDR corrected p-value with the edgeR DESeq2 edgeR DESeq2 2011 genome annotation gene8176 hypothetical protein DAPPUDRAFT_104167 104167* −6.24 −6.32 5.82E-44 4.45E-39 gene17246 cell recognition protein caspr4 isoform 5 109980* −5.05 −5.13 2.07E-07 2.51E-07 gene8175 hypothetical protein DAPPUDRAFT_225009 225009* −4.35 −4.41 4.49E-32 2.17E-28 gene5445 hypothetical protein DAPPUDRAFT_313428 313428* 4.98 4.97 8.54E-10 1.49E-17 gene3837 hypothetical protein DAPPUDRAFT_222529 222529* 6.92 6.91 5.40E-09 1.62E-20 gene16955 rna polymerase ii degradation factor 1-like 101472* 7.25 7.41 3.66E-16 1.41E-12 gene7919 hypothetical protein DAPPUDRAFT_324898 ---NA--- −5.73 −6.11 3.47E-04 1.83E-03 gene7984 metallothionein b 290503 4.09 4.07 2.83E-59 5.07E-132 gene6693 alpha-carbonic anhydrase ---NA--- 4.08 4.08 4.18E-03 3.41E-05 gene7509 hypothetical protein DAPPUDRAFT_335675 ---NA--- 4.16 4.14 9.39E-11 3.37E-18 gene6523 ---NA------NA--- 4.03 4.30 1.05E-06 1.65E-04 gene3259 hypothetical protein DAPPUDRAFT_211890 ---NA--- 4.17 4.16 2.33E-03 1.05E-05 gene17951 hypothetical protein DAPPUDRAFT_313929 ---NA--- 4.23 4.23 1.76E-06 3.88E-11 gene8356 hypothetical protein DAPPUDRAFT_106354 ---NA--- 4.44 4.47 3.05E-03 1.97E-05 gene5796 hypothetical protein DAPPUDRAFT_229695 ---NA--- 4.98 4.99 1.67E-03 4.49E-06 gene9671 hypothetical protein DAPPUDRAFT_333097 333097 5.10 5.10 1.39E-05 1.12E-10 gene12057 hypothetical protein DAPPUDRAFT_222523 ---NA--- 5.39 5.54 3.92E-08 9.87E-07 gene5697 hypothetical protein DAPPUDRAFT_337500 337500 6.75 7.03 1.06E-30 1.85E-13 gene2964 hypothetical protein DAPPUDRAFT_244715 ---NA--- 7.28 6.70 1.60E-04 1.30E-05 gene1954 vitelline membrane outer layer protein 1-like ---NA--- 8.68 8.85 1.07E-04 1.30E-07

Differential splicing events in response to acute copper genes involve conserved domain superfamily clusters re- exposure lated to functions that promote the insertion of copper Comparisons of the alternative splicing events between into cytochrome c oxidases (COX16), cellular detoxifica- all 15 copper-exposed samples with all 6 control samples tion (GST-C family), and anion translocation across identified 16 significantly differentially spliced (DS) membranes (ArsB NhaD permease; Table S5). Among genes (FDR corrected p-value < 0.05) with a difference in the DS genes, ES was the most abundant splicing type (6 exon inclusion level greater than 20% (Table 3). Func- genes) followed by A3SS (5 genes), A5SS (4 genes), MXE tional annotations of these genes included ion binding, (2 genes) and IR (1 gene) (Table 3; Table S5). Three DS DNA binding, transcription regulation, transmembrane genes – glutathione s-transferase (gst), alpha-aspartyl di- transport, signal transduction, metabolism, protein ubi- peptidase (pepe), and transmembrane protein 189 quitination, serine-type endopeptidase activity and pro- (tmem189) – were also found to be differentially teolysis. The alternatively spliced exons in 5 of these DS expressed.

Table 2 Distribution of alternative splicing (AS) types among the three clonal lineages AS Clone D Clone S Clone K Total Type No. of genes percentage No. of genes percentage No. of genes percentage A3SS 2477 52.5 2808 59.3 2669 55.4 4174 A5SS 2398 50.8 2143 45.2 2303 47.8 3796 MXE 104 2.2 111 2.3 115 2.4 129 ES 906 19.2 879 18.5 940 19.5 1181 IR 1058 22.4 1120 23.6 1076 22.3 1666 Suresh et al. BMC Genomics (2020) 21:433 Page 6 of 14

Table 3 Genes differentially spliced in response to acute Cu exposure from the global analysis combining all 6 controls with all 15 Cu exposed samples. Δψ is the absolute value of the difference in exon/intron inclusion levels between controls and Cu exposed samples Gene ID Description GO Term AS Spliced region |Δψ| type gene15738 coatomer subunit gamma binding A3SS Exon 3 0.255 gene7414 lethal malignant brain tumor-like protein 3 isoform ×2 transcription regulation; zinc ion A3SS Exon 7 0.342 binding gene16738 sialin isoform ×2 transmembrane transport A3SS Exon 8 0.291 gene4489 sprouty- evh1 domain-containing protein partial signal transduction A3SS Exon 4 0.293 gene8923 hypothetical protein DAPPUDRAFT_267459 metabolism A3SS Exon 3 0.281 gene7598 hypothetical protein DAPPUDRAFT_309480 single-stranded DNA binding A5SS Exon 1 0.302 gene9075 nucleoside diphosphate kinase 7 metabolism A5SS Exon 1 0.211 gene9075 nucleoside diphosphate kinase 7 metabolism ES Exon 1 partial 0.244 gene11416 protein grainyhead isoform ×1 NA A5SS Exon 12 0.346 gene5664 type i procollagen alpha 1 chain NA A5SS Exon 4 0.202 gene1314 glutathione s-transferase protein binding MXE Exon 4 and 9; Exon 5 and 0.267 10 gene8790 i’m not dead yet transmembrane transport MXE Exon 11 and 12 0.301 gene8790 i’m not dead yet transmembrane transport ES Exon 12 0.324 gene14576 pyruvate kinase-like isoform x ion binding; metabolism ES Exon 9 0.204 gene7153 tgf-beta-activated kinase 1 and map 3 k7-binding zinc ion binding ES 5′ UTR 0.278 protein 2 gene377 alpha-aspartyl dipeptidase serine-type peptidase activity; ES Exon 2 0.231 proteolysis gene10569 endophilin-a isoform ×1 protein binding; endocytosis ES Exon 8 0.213 gene15982 transmembrane protein 189 protein ubiquitination IR Intron 3 0.286

Lineage-specific patterns of gene expression and splicing consistently play a significant role in mediating tran- To investigate the influence of genetic background on scriptional response to acute Cu exposure in Daphnia the molecular response to acute copper exposure, the regardless of genetic background. gene expression and splicing patterns were compared In contrast to DE genes, there was very little overlap between controls (n = 2) and copper-exposed samples in DS genes among lineages: a total of 68 DS genes were (n = 5) of each clonal lineage. A total of 82 DE genes identified in Clone S, 101 DS genes in Clone D, and 65 were identified in Clone D, 119 DE genes in Clone K DS genes in Clone K (FDR corrected p-value < 0.05 and and 66 DE genes in Clone S (FDR corrected p-value < a difference in exon inclusion level greater than 20%), 0.05; Table S6). Of these, 6 DE genes were unique to out of which 56 were unique to Clone S, 82 were unique Clone D, 16 were unique to Clone K and 9 were unique to Clone D and 39 were unique to Clone K (Fig. 2b). to Clone S. Over 80% of the DE genes from clone- The most common differential splicing type observed in specific analyses overlapped with DE genes from the glo- all clones was exon skipping followed by use of alterna- bal analysis that combined all clonal lineages, suggesting tive 3′ splice site and use of alternative 5′ splice site that responses to acute Cu exposure are generally shared (Fig. 3; Tables S8,S9,S10). One gene (gene14576; pyru- among all clones (Fig. 2a). This is consistent with our vate kinase-like (pk) isoform x) was found to be differen- previous results reported in Chain et al. [53] using the tially spliced in all comparisons (including in each clone same dataset but a different reference assembly. Metallo- separately), suggesting that it plays an important role in thionein b (mtb), lipase 3 (lip3), rna polymerase ii deg- post-transcriptional Cu stress response in all clones re- radation factor 1 (def1), urea transporter 1-like gardless of the genetic background. Exon 9 of this gene (slc14A1), multiple inositol polyphosphate phosphatase 1 was skipped in samples exposed to Cu (Fig. 4), but the (minpp1) and eight other genes with unknown functions effect of this alternate transcript on protein function is were commonly differentially expressed in the clone- unknown and no conserved domains were found over- specific analysis and in the analysis combining all clonal lapping this exon (Table S5). Interestingly, Clone K, lineages (Table S7). This suggests that these genes which came from a copper-contaminated lake, had the Suresh et al. BMC Genomics (2020) 21:433 Page 7 of 14

Fig. 2 Venn diagram showing the overlap of differentially expressed and differentially spliced genes. Venn diagrams show the overlap of genes that display (a) differential expression (DE) or (b) differential splicing (DS) in the global analysis of all clones and the three separate analyses of clonal lineages. DE and DS analysis was carried out by grouping all clonal populations together (6 control samples vs 15 copper samples) or separately for each individual clone (2 control samples vs 5 copper samples) least number of DS genes but the highest number of DE only five GO terms remained enriched among upregu- genes. lated genes - proteolysis, serine-type endopeptidase activity, chitin binding, chitin metabolic process and Gene ontology (GO) enrichment analysis of DE and DS metallocarboxypeptidase activity; all these GO terms genes were also reported to be significantly enriched among A total of 45 Gene Ontology (GO) terms mostly belong- the upregulated DE genes in our previous analyses [53]. ing to 11 major functional categories were enriched Only one functional category was enriched among the among the upregulated DE genes, and 20 GO terms be- downregulated genes after FDR correction, extracellular longing to 10 major functional categories were enriched matrix structural constituent, which was also identified among the downregulated DE genes (weighted p-value to be enriched in our previous analyses [53]. Before any < 0.05; Table S11). After applying an FDR correction, FDR correction, enrichment analysis of DE genes from

Fig. 3 Number of differentially spliced (DS) genes according to splicing type. The DS splicing type is shown for Clones D, S and K in response to acute copper exposure. A3SS – alternate 3 prime splice site; A5SS – alternate 5 prime splice site; MXE – mutually exclusive exons; ES – exon skipping; IR – intron retention Suresh et al. BMC Genomics (2020) 21:433 Page 8 of 14

Fig. 4 Differential splicing of gene14576 (pyruvate kinase-like isoform x). The x-axis represents the exons and the y-axis represents the read coverage. The curved lines indicate splicing. Skipping of exon 9 is observed only in individuals exposed to copper (supported by an average of 9 reads across all 15 Cu exposed samples). Note that the y-axis scale is different in copper and control clone-specific analyses identified numerous enriched GO (82/6) followed by Clone S (56/9) and Clone K (39/16). terms that mostly overlap with the global analysis, in- This suggests that the splicing patterns induced by Cu cluding the five functions significant after FDR correc- are more variable between genetic lineages than gene ex- tion (Table S12). Because the topGO enrichment pression levels. The functional categories of metabolism, analysis algorithms already account for gene ontology catalytic activity, binding and transport were commonly topology, the topGO authors do not necessarily recom- enriched among the DE and DS genes in both the global mend the conservative FDR correction [54]. analyses and all three clone-specific analyses suggesting Among the DS genes, a total of 16 GO terms belong- that these pathways are regulated both at the level of ing broadly to the five functional categories of metabol- transcription and splicing in response to Cu. Certain ism, binding, catalytic activity, carbon utilization and functions such as signal transduction, stress response transport were enriched (weighted p-value < 0.05) when and cellular component organization were enriched all clones are combined (6 controls and 15 Cu-exposed among the DE but not the DS genes in the global ana- samples; Table S13). These same major categories plus a lyses, but were enriched among the DS genes in the few more were also enriched among the clones; Clones clone-specific analyses. For example, signal transduction D, S and K had 36 (7 functional categories), 25 (8 func- is enriched among the DE genes but only enriched tional categories) and 38 (9 functional categories) GO among the DS genes in Clones D and K. Similarly, stress terms enriched, respectively (weighted p-value < 0.05; response is enriched among the DE genes but only Fig. 5; Table S14). among the DS genes in Clones S and K. Cellular component analysis is enriched among the DS genes in all the Comparison between transcriptional and splicing three clones, but it is only enriched among the DE genes responses to acute Cu exposure in Daphnia pulex in Clone K. This suggests that some key pathways in- We found a much higher number of genes undergoing volved in response to metal exposure are jointly regu- differential expression than differential splicing in related by gene expression and splicing, but genetic sponse to acute Cu exposure from the global compari- variation between the clonal lineages also influences the son of all clones together: on average, 3.6% of all genes molecular responses observed. expressed across the 21 samples were differentially expressed compared to differential splicing of only 0.7% Discussion of the genes with alternative transcripts (0.1% of the Alternative splicing is an important regulatory mechan- expressed genes). However, from the clone-specific ana- ism that increases transcriptome and proteome diversity lyses, each independent clone has a higher number of and could potentially play an important role in mediat- unique DS genes than unique DE genes, with Clone D ing response to stress in addition to gene expression having the highest proportion of unique DS vs DE genes changes. Most studies investigating the molecular Suresh et al. BMC Genomics (2020) 21:433 Page 9 of 14

Fig. 5 Functional enrichment of differentially spliced genes. Distribution of enriched gene ontology (GO) terms for differentially spliced genes from the global analysis of all clones grouped together as well as from each individual clone analysis. The size of the circles is proportional to the number of observed genes within each GO category and the shade of the circles is proportional to the significance (measured in terms of the weighted p-value reported by topGO) mechanisms of response to environmental stressors have digestion, immune response, ion binding and trans- focused on gene expression responses without consider- port, signal transduction, chitin binding, exoskeletal ing the role of alternative splicing. To our knowledge, protein metabolism and chitinase activity, oxidative this is the first study investigating genome wide changes stress response and metal detoxification were signifi- in alternative splicing patterns in D. pulex. By comparing cantly differentially expressed [53, 55, 56]. Most of both the gene expression and splicing patterns in re- the DE genes identified in our previous study [53]were sponse to acute copper exposure in three D. pulex line- also detected in our current study, and the enriched func- ages, we provide evidence that alternative splicing is tional categories were almost identical. It is however not- prevalent and highly variable among lineages in response able that our analysis using the most recent genome to acute Cu stress. assembly detected almost three times as many DE genes, To investigate the molecular responses to acute suggesting an improved ability to detect differential gene copper exposure, we performed new analyses on our expression when a better-assembled genome is used. Out previously published dataset on gene expression in D. of the 20 genes that were found to have a 16-fold change pulex [53]. Our previous study focused on gene ex- in expression in copper-exposed samples compared to pression differences rather than differential splicing controls, only 6 were also reported to be strongly respon- response to acute Cu exposure, and also utilized an sive to Cu exposure in our previous study using the older older genome reference that is reported to have as- reference genome assembly [53]. Follow-up functional sembly errors leading to an overestimation of gene studies are needed to confirm this and to understand their number [42]. However, consistent with our previous potential role in mediating response to Cu stress in study, we found that genes involved in metabolism, Daphnia. Suresh et al. BMC Genomics (2020) 21:433 Page 10 of 14

Differential splicing response to acute Cu exposure cope with Cu stress, but could also be the result of per- Over 30% of the expressed genes across all 21 samples turbations of the splicing machinery induced by Cu [62]. underwent alternative splicing, and 16 genes were differ- The relative abundance of a specific transcript isoform entially spliced in response to copper when comparing of a gene depends on three factors: (a) the rate of tran- all 15 copper-exposed samples to the 6 controls. The scription of the gene, (b) the rate of splicing of the pri- gene pyruvate kinase-like (pk) isoform x (gene14576) was mary transcript to produce the specific transcript identified to be significantly differentially spliced both in isoform, and (c) a combination of both [63]. Therefore, the global analysis and in all three clones individually. changes in the splicing ratios of exons/introns can result The enzyme pyruvate kinase (PK) catalyzes the final re- in changes in the transcript abundances even without action of glycolysis and is known to have 4 isoforms in any change in overall gene expression. Alternative spli- mammals (M1, M2, R and L). The M1 and M2 isoforms, cing plays an important role in expanding proteome di- which are derived from alternative splicing of the same versity, and higher phenotypic complexity of eukaryotes gene (pyruvate kinase M - pkm), are identical except for has been attributed to a high frequency of AS events. a 160 bp section within the coding region [57]. The ac- Previous studies exploring the extent of gene expression tivity of PK is known to be inhibited by oxidative stress and splicing conservation across vertebrate species have in species ranging from bacteria to humans. In S. cerevi- reported that although alternative splicing is conserved siae, low PK activity resulting from oxidative stress has in a subset of orthologous exons and in certain tissues been associated with increased respiration which sup- such as brain and heart, alternative splicing diverges presses the production of reactive oxygen species [58]. more rapidly between species than gene expression [64, In D. pulex, this gene consists of 11 exons and has two 65]. Species-specific differences in alternative splicing mRNA isoforms in both controls and Cu-exposed sam- patterns were also shown to have a more profound effect ples. One of the isoforms consists of exons 1 to 9 while on pharyngeal jaw diversification in Lake Tanganyika the other isoform consists of exons 1–7 and 10–11. Here cichlids than differences in gene expression, indicating we found differential splicing where exon 9 was skipped that alternative splicing plays an important role in the only in samples exposed to Cu, suggesting the presence evolution of distinct morphologies in cichlid adaptive ra- of a shorter transcript isoform of this gene expressed diation [66]. This highlights that at least among verte- only in response to Cu exposure (Fig. 4). Further investi- brates, alternative splicing is a potentially powerful gation of the functional role of this additional mRNA mechanism shaping species-specific differences. Differ- lacking exon 9 can help understand whether it has a role ences in alternative splicing patterns between species are in mitigating Cu-induced oxidative stress in Daphnia. possibly caused by changes in the cis-regulatory ele- Additionally, there is one exon in the two genes nucleo- ments (such as splicing enhancers or silencers) or trans- side diphosphate kinase 7 (ndk7; gene 9075) and i’m not acting factors [65]. The three clonal lineages used in this dead yet (indy; gene 8970) that is involved in multiple study have had different Cu exposure histories, which splicing events. While it is possible for one gene to have might have led to differential selection of genotypes re- multiple transcript isoforms, further investigation is related to Cu tolerance in their originating natural popula- quired to understand the exact splicing mechanism of tions [53] and differences in cis-regulatory elements or these genes and the putative functional role of the alter- trans-acting factors. Thus, differences in splicing pat- native transcripts in relation to Cu stress. terns among the clones could possibly be linked to their historical exposure to Cu and differences in Cu Clone-specific patterns of differential splicing sensitivity. To investigate whether different genotypes of D. pulex Genes related to metabolism, transport, binding, cata- vary in their response to acute copper exposure, we ana- lytic activity, oxidative stress response, metal homeosta- lyzed patterns of gene expression and splicing separately sis and detoxification were commonly DE and DS across in each clone. Interclonal variation in heavy metal sensi- all three clonal lineages. Previous studies on Daphnia in- tivity has been shown in plants [59, 60] and previous vestigating transcriptional response to heavy metal tox- studies on Daphnia have reported that molecular re- icity have reported differential expression of genes sponse to heavy metals varied across genotypes [49, 53, involved in these pathways suggesting that these are key 61]. Gene expression response to acute Cu stress was pathways associated with response to metal toxicity [53, more similar among the three clones compared to spli- 55, 67]. The fact that we find the genes involved in these cing. The three clones have on average 7.4 times more pathways to also be DS indicates that they are regulated unique DS genes than unique DE genes, suggesting that via transcription and splicing. In contrast, certain func- genetic background has a larger effect on differential tional pathways were only enriched either among the DE splicing than on differential expression. These intraspe- or DS genes. The genes related to exoskeletal protein cific differences could reflect diverse pathways used to metabolism and immune response were only enriched Suresh et al. BMC Genomics (2020) 21:433 Page 11 of 14

among the DE genes whereas the genes related to post- copper exposure in D. pulex [53] (study accession translational modification (PTM) were only enriched PRJEB28650 in the ENA database). This transcriptomic among the DS genes. In addition to regulating the ex- dataset consisted of a total of 6 control samples and 15 pression of genes in response to stressors, PTM of exist- copper exposed samples from three different clonal line- ing proteins can help in mitigating the effects of the ages (Fig. 1a). Briefly, one D. pulex individual was iso- stressor and restoring cellular homeostasis especially lated from each of three habitats varying in historical after transient exposure [68]. Exposure of yeast to in- copper contamination levels and cultured in the labora- creased levels of Cu results in proteolysis of the copper tory for about a year to establish the three clonal line- transport gene ctr1, and exposure to Zn causes ubiquiti- ages (D, S and K). Toxicity assays were conducted in the nation of the Zn transporter gene zrt1, thereby prevent- lab to evaluate the relative Cu tolerance of each clone. ing further uptake of Cu or Zn respectively [69, 70]. Two clones (D and S) are from habitats not known to be Thus, post-translational control provides a fast-acting al- contaminated with heavy metals, whereas the other ternative approach to mitigating Cu toxicity. To our clone (K) is from a lake contaminated with high levels of knowledge, none of the previous studies on Daphnia in- Cu (100 μg/L) as well as Ni and Mn [53, 71]. The clones vestigating gene expression response to metal toxicity were subjected to an acute Cu exposure experiment for have reported differential expression of genes involved gene expression analysis, in which five replicates of each in the PTM process suggesting that the involvement of clone were separated into different tanks and either ex- these genes in response to Cu stress occurs solely via posed to 90 μg/L Cu for 24 h or raised in a controlled splicing. environment without Cu. Experiments were conducted in FLAMES medium [72] at 18 degrees Celsius under a Conclusion 16:8 light-dark cycle, and were fed 20,000 cells of algae Our findings suggest that genetic variation has a large per mL [53]. After the exposure experiment, two repli- impact on molecular responses to acute Cu exposure in cates from each clone in control conditions were se- D. pulex. A limitation of our study is that it included lected for RNA extraction, as well as five replicates from only two control replicates per clonal lineage, and hence each clone in Cu conditions (Fig. 1a). Total RNA was future studies with larger sample sizes investigating the extracted from whole primiparous adult Daphnia, result- influence of interclonal variation on gene expression re- ing in 21 RNA-seq libraries that were sequenced on two sponse to metal stress would be needed to validate our lanes of an Illumina HiSeq 2000 (100 bp paired-end findings. In addition, it is possible that we underesti- reads) at Genome Quebec. mated splicing events of lowly expressed genes or those with few reads spanning splice junctions. While we Bioinformatics found certain functions involved in Cu stress response The raw sequence data of each of the 21 samples were to be common across all clones, there was variation in checked for sequencing quality using FastQC [73]. Low Cu stress-response pathways among the clones. As the quality sequences were trimmed using Trimmomatic lineages in our study varied in their prior exposure to (ILLUMINACLIP:TruSeq3-PE.fa:2:30:15:8:true TRAIL- sub-lethal levels of Cu, acclimation or adaptation to an ING:30 MINLEN:90 CROP:90) [74]. All sequences were environment with high Cu levels in the wild could have trimmed to an equal length of 90 bp. Using STAR [75], influenced the level of variation found in the splicing re- the sequences were then mapped to a recently revised D. sults, if this effect persists for many generations after pulex reference genome (assembly PA42 3.0) [42]. The culturing in the lab in the absence of Cu. Alternatively, resulting BAM files along with the reference annotation high levels of variation could be driven by perturbations file obtained from [42] were submitted to featureCounts of the splicing machinery by copper stress. By comparing [76] to determine raw read counts per gene. Differential the transcriptional and splicing response to acute Cu ex- expression analysis was performed between all the posure, we conclude that interclonal variation can have copper-exposed (n = 15) and control samples (n =6) a substantial influence on splicing and argues for an in- using DESeq2 v1.22.2 [77] and edgeR v3.24.3 [78]. The crease in attention to alternative splicing in toxicoge- genes were considered to be differentially expressed nomic and gene expression studies. (DE) between the two conditions if the False Discovery Rate (FDR) adjusted p-value was less than 0.05 in both Methods DESeq2 and edgeR analyses. A reciprocal BLAST ap- Gene expression dataset proach was used with default parameters in BLASTn To address the effects of copper on differential gene ex- [79] to determine if the DE genes identified in this study pression and alternative splicing, a gene expression data- matched those identified in our previous study as re- set was acquired from our recent experiment ported in Chain et al. [53], in which we used the same investigating transcriptional consequences of acute transcriptome data but a different reference genome and Suresh et al. BMC Genomics (2020) 21:433 Page 12 of 14

annotation [41]. For each gene, only BLAST hits that et al. 2017 [42]. Table S4. Comparison of the DE genes identified by had an E-value <1e-10 were considered, and the two top Chain et al. 2019 [53] and the genes from the newer Daphnia PA42 3.0 BLAST hits were considered if they were within 10-bit reference assembly from Ye et al. 2017 [42] using BLASTn. Table S5. Dif- ferentially spliced genes in response to acute Cu exposure when compar- scores of one another. ing all 6 controls with all 15 Cu exposed samples. Δ|Ψ| is the difference Identification of alternative splicing (AS) events and in the exon/intron inclusion level between the two conditions. Table S6. differential splicing (DS) analysis was conducted using Differentially expressed genes in response to acute Cu exposure from clone specific analyses. Table S7. Genes commonly differentially rMATS v3.2.5 [25], which uses reads spanning both expressed from global analysis and clone specific analyses. Table S8. Dif- splice junctions and exons. The splice junctions for all ferentially spliced genes in response to acute Cu exposure in Clone D. identified alternative splicing events were required to be Table S9. Differentially spliced genes in response to acute Cu exposure in Clone S. Table S10. Differentially spliced genes in response to acute supported by at least five uniquely mapped reads and Cu exposure in Clone K. Table S11. Gene Ontology enrichment analysis additionally have a minimum of 10 nt overhang for of the DE genes from the global analysis combining all 6 controls with all A3SS, A5SS, ES and MXE in at least one sample in each 15 Cu exposed samples (BP: biological process, MF: molecular function). Table S12. Gene Ontology enrichment analysis of the DE genes from condition. The genes were considered to be differentially clone specific analyses (BP: biological process, MF: molecular function). spliced (DS) if the adjusted p-value after Benjamini- Table S13. Gene Ontology enrichment analysis of the DS genes from Hochberg correction was less than 0.05 and if the differ- the global analysis combining all 6 controls with all 15 Cu exposed sam- Δ ψ ples (BP: biological process, MF: molecular function). Table S14. Gene ence in exon inclusion levels ( | |) was greater than Ontology enrichment analysis of the DS genes from clone specific ana- 20%. To determine whether the clonal lineages respond lyses (BP: biological process, MF: molecular function). differently to Cu stress, differential gene expression and differential splicing analysis was also carried out between Abbreviations the copper-exposed (n = 5) and control samples (n =2) A3SS: Alternative 3′ splice site; A5SS: Alternative 5′ splice site; AS: Alternative separately for each clone. While clone-specific analyses splicing; Cu: Copper; DE: Differential expression; DS: Differential splicing; ES: Exon skipping; GO: Gene ontology; IR: Intron retention; mRNA: Messenger have lower statistical power to detect global effects of RNA; MXE: Mutually exclusive exon; PK: Pyruvate kinase; PSI: Percent spliced copper-exposure due to lower sample sizes, they can re- in; PTM: Post translational modification veal lineage-specific differences that would not be detected from the global analysis. Splicing plots were Acknowledgements We thank Dr. Sen Xu and Dr. Michael Lynch for providing GO annotations generated using ggsashimi [80]. for the Daphnia PA42.3.0 genome assembly. To identify potential biological pathways involved in response to acute Cu exposure, Gene Ontology (GO) en- Authors’ contributions richment was performed for the DE and DS genes with SS conducted the analyses and prepared the figures. SS and FC designed the methods and drafted the manuscript. MC and TC designed the copper topGO v2.30.1 [81] using the default algorithm exposure experiment. All authors contributed to interpreting the data and (weight01). This tests for enrichment of particular func- editing the manuscript. The author(s) read and approved the final tional categories among the list of DS genes compared manuscript. to all expressed genes in our samples. GO terms with a Funding weighted p-value less than 0.05 were considered to be This project was partly supported by an NSERC CREATE grant (397997–11) on significantly enriched. The sequences of alternatively Aquatic Ecosystem Health to MC, and startup funds from UMass Lowell to spliced exons were also used in searches against the FC. The funding sources played no role in the design of the study, data collection, analysis, interpretation, or writing of the manuscript. NCBI conserved domains database to identify putative protein domains involved in alternative splicing. Availability of data and materials The dataset used and analyzed in the current study is available from the ENA database under study accession PRJEB28650. Supplementary information Supplementary information accompanies this paper at https://doi.org/10. Ethics approval and consent to participate 1186/s12864-020-06831-4. Not applicable.

Additional file 1: Table S1. Quality and mapping information for the Consent for publication 21 Daphnia pulex RNA-sequencing libraries against both the JGI V1.0 Not applicable. 2011 assembly from Ensembl (as reported in Table S3 in Chain et al. 2019 [53]) and also against the newer Daphnia PA42 3.0 reference assembly Competing interests from Ye et al. 2017 [42]. Sample names consist of tank number (T), individual ID number, and clone (D, K or S). Samples were run in one of two The authors declare that they have no competing interests. lanes, and average quality (Q) is presented as PHRED scores. The % mapped reads and number of expressed genes are shown for each of Author details 1Department of Biological Sciences, University of Massachusetts Lowell, the reference assemblies, as well as the alternative spliced genes against 2 the newer reference. Table S2. Differentially expressed genes in response Lowell, MA 01854, USA. Present address: The Swire Institute of Marine Science and School of Biological Sciences, The University of Hong Kong, to acute Cu exposure when comparing all 6 controls with all 15 Cu ex- 3 posed samples. Table S3. Summary of blast analysis comparing the dif- Pokfulam Road, Pok Fu Lam, Hong Kong SAR. Department of Integrative Biology, University of Guelph, Guelph, ON N1G 2W1, Canada. 4Department of ferentially expressed (DE) genes identified by Chain et al. [53] with the genes from the newer Daphnia PA42 3.0 reference assembly from Ye Biology, McGill University, 1205 Docteur Penfield, Montreal, QC H3A 1B1, Canada. Suresh et al. BMC Genomics (2020) 21:433 Page 13 of 14

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